For each chromosome to properly segregate during mitosis, its kinetochores must bipolarly attach spindle microtubules. The failure of chromosomes to biorient is a major cause of cellular aneuploidy, a driving force in cancer and birth defects. Bipolar attachment is achieved because tension is produced between sister kinetochores, which both stabilizes microtubule attachments and turns off spindle checkpoint signals. A key to understanding how cells become aneuploid is to understand how chromosomes sense tension between sister kinetochores and use this to regulate microtubule attachment and spindle checkpoint signals. Proteins that localize to the inner centromere are central to these processes and these proteins form a network to regulate the Aurora B kinase which is a member of the chromosome passenger complex. We have purified the CPC to homogeneity and developed a system to study its activation in vitro. These experiments are uncovering both positive and negative feedback loops as well as the key mutants to dissect the role of these pathways in vivo. To characterize mutants we are employing the animal caps of Xenopus embryos which allow us to easily knockdown and replace proteins and dissect phenotypes in normal diploid tissue. The combination of in vitro biochemistry, Xenopus extracts and now dissection of phenotypes in animal caps provides a unique opportunity to move seamlessly between biochemical and cell biological approaches in a vertebrate system. We hypothesize that one role of the CPC is to generate gradients of soluble phosphoactivity that provide spatial information to pattern the 3D space of the cell for mitotic events. We will also test this important hypothesis as well as determine the role of Aurora B in generating a central band of RhoA that determines the location of the cytokinetic furrow. Finally we will perform purification of inner centromere chromatin to systematically identify proteins that localize to this chromosome territory as well as the DNA sequences that they are assembled upon.